1. Field of the Invention
The present invention relates to a process for the production of herbicide-tolerant plants by expressing an exogenous herbicide-binding polypeptide in plants or plant organs. The invention furthermore relates to the use of the corresponding nucleic acids which encode a polypeptide, an antibody or parts of an antibody with herbicide-binding properties in transgenic plants, and the thus transformed plant itself.
2. Description of the Related Art
It is known that genetic engineering methods allow the specific transfer of foreign genes into the genome of a plant. This process is termed transformation, and the resulting plants transgenic plants. Transgenic plants are currently being employed in various fields of biotechnology. Examples of insect-resistant plants (Vaek et al. Plant Cell 5 (1987), 159-169), virus-resistant plants (Powell et al. Science 232 (1986), 738-743) and ozone-resistant plants (van Camp et al. BioTech. 12 (1994), 165-168). Examples of improved quality characteristics achieved by genetic engineering are: improved shelf life of fruit (Oeller et al. Science 254 (1991), 437-439), increased starch production in potato tubers (Stark et al. Science 242 (1992), 419), changes in starch (Visser et al. Mol. Gen. Genet. 225 (1991), 289-296) and lipid composition (Voelker et al. Science 257 (1992), 72-74), and production of foreign polymers (Poirer et al. Science 256 (1992), 520-523).
An important target of work carried out in the field of plant molecular genetics is the generation of herbicide tolerance. Herbicide tolerance is characterized by an improved compatibility (in terms of type or level) of the plant or plant organs with the herbicide applied. This can be effected in various ways. The known methods are utilization of a metabolic gene, for example the pat gene, in connection with glufosinate resistance (WO 8705629) or a target enzyme which is resistant to the herbicide, such as in the case of enolpyruvyl shikimate-3-phosphate synthase (WO 9204449), which is resistant to glyphosate, and the use of a herbicide in cell and tissue culture for the selection of tolerant plant cells and resulting resistant plants, such as described in the case of acetyl-CoA-carboxylase inhibitors (U.S. Pat. No. 5,162,602, U.S. Pat. No. 5,290,696).
Antibodies are proteins as component of the immune system. A joint feature of all antibodies is their spatial, globular structure, the construction of light and heavy chain and their basic capability of binding molecules or parts of a molecular structure with high specificity (Alberts et al., in: Molekularbiologie der Zelle [Molecular Biology of the Cell], 2nd Edition 1990, VCH Verlag, ISBN 3-527-27983-0, 1198-1237). On the basis of these properties, antibodies have been utilized for a number of tasks. Application can be divided into application of the antibodies within the animal and human organisms in which they are produced, that is to say the so-called in-situ applications, and the ex-situ applications, ie. utilization of the antibodies after they have been isolated from the producing cells or organisms (Whitelam und Cockburn, TIPS Vol.1, 8 (1996), 268-272).
The use of somatic hybrid cell lines (hybridomas) as a source of antibodies against very specific antigens is based on work carried out by Kxc3x6hler and Milstein (Nature 256 (1975) 495-97). This process allows so-called monoclonal antibodies to be produced which have a uniform structure and which are produced by means of cell fusion. Spleen cells of an immunized mouse are fused with mouse myeloma cells. This gives hybridoma cells which proliferate infinitely. At the same time, the cells secrete specific antibodies against the antigen with which the mouse had been immunized. The spleen cells provide the capability of antibody production while the myeloma cells contribute the capacity of unlimited growth and continuous secretion of antibodies. Since each hybridoma cell, being a clone, is derived from a single B cell, all antibody molecules produced have the same structure, including the antigen binding site. This method has greatly promoted the use of antibodies since antibodies which have a single, known specificity and a homogeneous structure are now available in unlimited quantities. Monoclonal antibodies are used widely in immunodiagnostics and as therapeutics.
In recent years, the so-called phage display method has become available for the production of antibodies, and here the immune system and the various immunizations in the animal are avoided. The affinity and specificity of the antibody are made to measure in vitro (Winter et al., Ann. Rev. Immunol. 12 (1994), 433-455; Hoogenboom TIBTech Vol 15 (1997), 62-70). Gene segments which contain the sequence which encodes the variable region of antibodies, ie. the antigen binding site, are fused with genes for the coat protein of a bacteriophage. Then, bacteria are infected with phages which contain such fusion genes. The resulting phage particles are now equipped with coats containing the antibody-like fusion protein, the antibody-binding domain pointing outward. Such a phage display library can now be used for isolating the phage which contains the desired antibody fragment and which binds specifically to a certain antigen. Each phage isolated in this manner produces a monoclonal antigen-binding polypeptide which corresponds to a monoclonal antibody. The genes for the antigen binding site, which are unique for each phage, can be isolated from the phage DNA and employed for constructing complete antibody genes.
In the field of crop protection, antibodies were utilized in particular as analytical tools ex-situ for the qualitative and quantitative detection of antigens. This includes the detection of plant constituents, herbicides or fungicides in drinking water (Sharp et al. (1991) ACS Symp Ser., 446 (Pestic. Residues Food Saf.) 87-95), soil samples (WO 9423018) or in plants or plant organs, and the utilization of antibodies as auxiliaries for the purification of bound molecules.
The production of immunoglobulins in plants was first described by Hiatt et al., Nature, 342 (1989), 76-78. The spectrum encompasses single-chain antibodies up to multimeric secretory antibodies (J. Ma and Mich Hein, 1996, Annuals New York Academy of Sciences, 72-81).
More recent attempts utilize antibodies in-situ for defending plants against pathogens, in particular viral diseases, by expressing, in plant cells, specific antibodies or parts thereof which are directed against viral coat proteins (Tavladoraki et al., Nature 366 (1993), 469-472; Voss et al., Mol. Breeding 1 (1995), 39-50).
An analogous approach has also been utilized for defending the plant against infection by nematodes (Rosso et al., Biochem Biophys Res Com, 220 (1996) 255-263). There exist examples for an application in pharmacology where the in-situ expression of antibodies in plants is utilized for oral immunization (Ma et al., Science 268 (1995), 716-719; Mason and Arntzen, Tibtech Vol 13 (1996), 388-392). The body is provided with antibodies formed by the plant and originating from plants or plant organs which are suitable for consumption, via the mouth, throat or digestive tract, which antibodies cause efficient immunoprotection. Moreover, a single-chain antibody against the low-molecular-weight plant hormone abscisic acid has already been expressed in plants, and a reduced availability of plant hormones, due to binding of abscisic acid in the plant, has been observed (Artsaenko et al., The Plant Journal 8 (5) (1995) 754-750).
Chemical control of weeds in agronomically important crops requires the use of highly selective herbicides. However, in some cases it is difficult to develop sufficiently selective herbicides which do not cause damage of the plant which provides the yield in any crop. The introduction of herbicide-resistant or -tolerant crop plants can contribute to solving this problem.
The development of herbicide-resistant crop plants by tissue culture or seed mutagenesis and natural selection is limited. Only those plants can be manipulated via tissue culture techniques where entire plants can be regenerated successfully from cell cultures. Moreover, following mutagenesis and selection, crop plants may display undesirable characteristics which have to be reeliminated by, in some cases repeated, back-crossing. Also, the introduction of a resistance by performing crosses would be restricted to plants of the same species.
It is for the abovementioned reasons that the genetic engineering approach of isolating a resistance-encoding gene and transferring it into crop plants in a targeted manner is superior to the traditional plant breeding method.
To date, the development of herbicide-tolerant or herbicide-resistant crop plants, by molecular biology methods, requires a knowledge of the mechanism of action of the herbicide in the plant and also that genes which impart resistance to the herbicide can be found. A large number of herbicides which are presently utilized commercially act by blocking an enzyme of an essential amino acid, lipid or pigment biosynthesis step. Herbicide tolerance can be generated by altering the genes of these enzymes in such a way that the herbicide can no longer be bound and by introducing these altered genes into crop plants. An alternative example is to find analogous enzymes in nature, for example in microorganisms which exhibit a natural resistance to the herbicide. This resistance-imparting gene is isolated from such a microorganism, recloned into suitable vectors and subsequently, after successful transformation, expressed in herbicide-sensitive crop plants (WO 96/38567).
It was an object of the present invention to develop a novel, generally utilizable genetic engineering method for producing herbicide-tolerant transgenic plants.
We have found that this object is achieved, surprisingly, by a process of expressing, in the plants, an exogenous polypeptide, antibody or parts of an antibody with herbicide-binding properties.
The present invention relates firstly to the production of a herbicide-binding antibody and the cloning of the relevant gene or gene fragment.
The first step is to produce a suitable antibody which binds the herbicide. This can be effected, inter alia, by immunizing a vertebrate, in most cases mouse, rat, dog, horse, donkey or goat, with an antigen. The antigen in this case is a fungicidally active compound which is associated or coupled to a higher-molecular-weight carrier such as bovine serum albumin (BSA), chicken ovalbumin, keyhole limpet hemocyanine (KLH) or other carriers, via a functional group. After antigen has been applied repeatedly, the immune response is monitored with customary methods, and a suitable antiserum is thus isolated. Initially, this approach yields a polyclonal serum which contains antibodies with differing specificities. For the targeted in-situ use, it is necessary to isolate the gene sequence which encodes a single, specific, monoclonal antibody. A variety of routes are available for this purpose. The first approach exploits the fusion of antibody-producing cells and cancer cells to give a hybridoma cell culture which continuously produces antibodies and which finally, by singling the clones obtained, leads to a homogeneous cell line which produces a defined monoclonal antibody.
The cDNA for the antibody, or parts of the antibody, viz. the so-called single chain antibody (scFv), is isolated from such a monoclonal cell line. These cDNA sequences can then be cloned into expression cassettes and used for the functional expression in prokaryotic and eukaryotic organisms, including plants.
Alternatively, it is possible to select antibodies via phage display libraries, and these antibodies bind herbicide molecules and convert them catalytically into a product which has non-fungicidal properties. Methods for raising catalytic antibodies are described in Janda et al., Science 275 (1997) 945-948, Chemical selection for catalysis in combinatorial Antibody libraries; Catalytic Antibodies, 1991, Ciba Foundation Symposium 159, Wiley-Interscience Publication. Cloning the gene of this catalytic antibody and expressing it in a plant may, in principle, also lead to a herbicide-resistant plant.
The invention particularly relates to expression cassettes whose encoding sequence encodes a herbicide-binding polypeptide or a functional equivalent thereof, and to the use of these expression cassettes for the production of a herbicide-tolerant plant. The nucleic acid sequence can be, for example, a DNA sequence or a cDNA sequence. Encoding sequences which are suitable for insertion into an expression cassette according to the invention are, for example, those which contain a DNA sequence from a hybridoma cell which encodes a polypeptide with herbicide-binding properties and thus impart resistance to plant-enzyme inhibitors to the host.
Moreover, the expression cassettes according to the invention contain regulatory nucleic acid sequences which govern expression of the encoding sequence in the host cell. In a preferred embodiment, an expression cassette according to the invention comprises upstream, ie. on the 5xe2x80x2-end of the encoding sequence, a promoter and downstream, ie. on the 3xe2x80x2-end, a polyadenylation signal and, if appropriate, other regulatory elements which are linked operatively with the in-between encoding sequence for the polypeptide with herbicide-binding properties and/or transit peptide. Operative linkage is to be understood as meaning the sequential arrangement of promoter, encoding sequence, terminator and, if appropriate, other regulatory elements in such a way that each of the regulatory elements can function as intended when the encoding sequence is expressed. The sequences preferred for operative linkage, but not limited thereto, are targeting sequences for guaranteeing subcellular localization in the apoplasts, in the plasma membrane, in the vacuole, in plastids, into the mitochondrium, in the endoplasmatic reticulum (ER), in the nucleus, in liposomes or in other compartments and translation enhancers, such as the 5xe2x80x2-leader sequence from the tobacco mosaic virus (Gallie et al., Nucl. Acids Res. 15 (1987) 8693-8711).
Suitable promotors [sic] of the expression cassette according to the invention is, in principle, any promoter which is capable of governing the expression of foreign genes. Promoters which are preferably used are, in particular, a plant-derived promoter or a promoter originating from a plant virus. Particularly preferred is the CaMV 35S promotor from the cauliflower mosaic virus (Franck et al., Cell 21(1980) 285-294). This promoter contains various recognition sequences for transcriptional effectors, which, in their totality, lead to permanent and constitutive expression of the gene introduced (Benfey et al., EMBO J. 8 (1989) 2195-2202).
The expression cassette according to the invention may also comprise a chemically inducible promoter by means of which expression of the exogenous polypeptide in the plant can be controlled at a particular point in time. Such promoters, for example the PRP1 promotor (Ward et al., Plant.Mol.Biol.22(1993), 361-366), a promoter which is inducible by salicylic acid (WO 95/1919443), a promoter which is inducible by benzenesulfonamide (EP 388186), a promoter which is inducible by abscisic acid (EP335528) or a promoter which is inducible by ethanol or cyclohexanone (WO9321334), have been described in the literature and can be used, among others.
Other promoters which are particularly preferred are those which guarantee expression in tissues or plant organs in which the herbicidal activity takes place. Promoters which guarantee leaf-specific expression deserve particular mention. Mention must be made of the potato cytosolic FBPase promoter or the potato ST-LSI promotor (Stockhaus et al., EMBO J. 8 (1989) 2445-245).
The stable expression of single-chain antibodies, which amounted to up to 0.67% of the total soluble seed protein in the seeds of transgenic tobacco plants, was made possible with the aid of a seed-specific promoter (Fiedler and Conrad, Bio/Technology 10(1995), 1090-1094). Since expression may also be possible in seeds which have been sown or which are in the process of germination and may be desired for the purposes of the present invention, such germination- and seed-specific promoters are also regulatory elements which are preferred in accordance with the invention. Thus, the expression cassette according to the invention can therefore contain, for example, a seed-specific promoter (preferably the USP or LEB4 promotor), the LEB4 signal peptide, the gene to be expressed, and an ER retention signal. The construction of the cassette is shown by way of example in the form of a diagram in FIG. 1 with reference to a single-chain antibody (scFv gene).
An expression cassette according to the invention is produced by fusing a suitable promoter with a suitable polypeptide DNA and, preferably, a DNA which encodes a chloroplast-specific transit peptide and which is inserted between promoter and polypeptide DNA, and a polyadenylation signal, using customary recombination and cloning techniques as they are described, for example, in T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) and also in T. J. Silhavy, M. L. Berman and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and in Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1987).
Particularly preferred are sequences which allow targeting into the apoplast, the plastids, the vacuole, into the plasma membrane, the mitochondrium, the endoplasmatic reticulum (ER) or, by the absence of suitable operative sequences, residence in the compartment of formation, namely the cytosol (Kermode, Crit. Rev. Plant Sci. 15, 4 (1996), 285-423). Localization in the ER and the cell wall have proved to be especially beneficial for quantitative protein accumulation in transgenic plants (Schouten et al. , Plant Mol. Biol. 30 (1996), 781-792; Artsaenko et al., Plant J. 8 (1995) 745-750).
The invention also relates to expression cassettes whose encoding sequence encodes a herbicide-binding fusion protein, part of the fusion protein being a transit peptide, which governs translocation of the polypeptide. Especially preferred are chloroplast-specific transit peptides which are cleaved enzymatically from the herbicide-binding polypeptide moiety after the herbicide-binding polypeptide has been translocated into the plant""s chloroplasts. Particularly preferred is the transit peptide derived from plastid transketolase (TK) or a functional equivalent of this transit peptide (for example the transit peptide of the small subunit of Rubisco or ferredoxin NADP oxidoreductase).
The polypeptide DNA or polypeptide cDNA required for the production of expression cassettes according to the invention is preferably amplified with the aid of polymerase chain reaction (PCR). DNA amplification methods using PCR are known, for example from Innis et al., PCR Protocols, A Guide to Methods and Applications, Academic Press (1990). The PCR-produced DNA fragments can expediently be checked by sequence analysis to avoid polymerase errors in constructs to be expressed.
The nucleotide sequence inserted, which encodes a herbicide-binding polypeptide, can be generated synthetically or obtained naturally or comprise a mixture of synthetic and natural DNA components. In general, synthetic nucleotide sequences with codons which are preferred by plants are prepared. These codons which are preferred by plants can be determined from codons whose proteins are most frequent and which are expressed in most of the interesting plant species. When preparing an expression cassette, various DNA fragments can be manipulated so as to obtain a nucleotide sequence which expediently reads in the correct sense and which is equipped with a correct reading frame. To connect the DNA fragments to each other, adaptors or linkers can be added to the fragments.
The promoter and terminator regions according to the invention should expediently be provided, in the sense of the transcription, with a linker or polylinker comprising one or more restriction sites for insertion of this sequence. As a rule, the linker has 1 to 10, usually 1 to 8, preferably 2 to 6, restriction sites. Within the regulatory regions, the linker generally has a size of less than 100 bp, frequently less than 60 bp, but at least 5 bp. The promoter according to the invention can be either native or homologous or else foreign or heterologous to the host plant. The expression cassette according to the invention comprises, in the 5xe2x80x2-3xe2x80x2-sense of transcription, the promoter according to the invention, any desired sequence and a region for transcriptional termination. Various termination regions are mutually exchangeable as desired.
Furthermore, manipulations which provide suitable restriction sites or which remove excess DNA or restriction sites can be employed. Where insertions, deletions or substitutions, for example transitions and transversions, are possible, in-vitro mutagenesis, xe2x80x9cprimer repairxe2x80x9d, restriction or ligation may be used. In the case of suitable manipulations such as restriction, xe2x80x9cchewing-backxe2x80x9d or filling up projections for xe2x80x9cblunt endsxe2x80x9d, complementary ends of the fragments may be provided for ligation purposes.
Especially important for the success according to the invention is the attachment of the specific ER retention signal SEKDEL (Schuoten, A. et al. Plant Mol. Biol. 30 (1996), 781-792), with which the average expression level is trebled to quadrupled. Other retention signals which occur naturally in plant and animal proteins which are localized in the ER may also be used for constructing the cassette.
Preferred polyadenylation signals are plant polyadenylation signals, preferably those which correspond essentially to T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular gene 3 of the T-DNA (octopin synthase) of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984) 835 et seq.) or functional equivalents.
An expression cassette according to the invention may comprise, for example, a constitutive promotor (preferably the CaMV 35 S promotor), the LeB4 signal peptide, the gene to be expressed and the ER retention signal. The construction of the cassette is shown as a diagram in FIG. 2 with reference to a single-chain antibody (scFv gene). The amino-acid sequence KDEL (lysine, aspartic acid, glutamic acid, leucine) is preferably used as ER retention signal.
The fused expression cassette which encodes a polypeptide with herbicide-binding properties is preferably cloned into a vector, for example pBin19, which is suitable for transforming Agrobacterium tumefaciens. Agrobacteria which are transformed with such a vector can then be used in the known manner for transforming plants, in particular crop plants, eg. tobacco plants, by, for example, bathing wounded leaves or leaf sections in an Agrobacterial solution and subsequently growing them in suitable media. The transformation of plants by means of Agrobacteria is known, inter alia, from F. F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S. D. Kung and R. Wu, Academic Press, 1993, pp. 15-38, and from S. B. Gelvin, Molecular Genetics of T-DNA Transfer from Agrobacterium to Plants, also in Transgenic Plants, pp. 49-78. Transgenic plants can be regenerated from the transformed cells of the wounded leaves or leaf sections in the known manner, and these transgenic plants contain a gene for the expression of a polypeptide with herbicide-binding properties, integrated into the expression cassette according to the invention.
To transform a host plant with a DNA encoding a herbicide-binding polypeptide, an expression cassette according to the invention is incorporated, as an insertion, into a recombinant vector whose vector DNA contains additional functional regulation signals, for example sequences for replication or integration. Suitable vectors are described, inter alia, in xe2x80x9cMethods in Plant Molecular Biology and Biotechnologyxe2x80x9d (CRC Press), (1993) chapter 6/7, pp.71-119.
Using the above-cited recombination and cloning techniques, the expression cassettes according to the invention can be cloned into suitable vectors which allow them to be multiplied, for example in E. coli. Suitable cloning vectors are, inter alia, pBR332, pUC series, M13mp series and pACYC184. Especially suitable are binary vectors which can replicate in both E. coli and agrobacteria, for example pBin19 (Bevan et al. (1980) Nucl. Acids Res. 12, 8711).
The invention furthermore relates to the use of an expression cassette according to the invention for the transformation of plants, plant cells, plant tissues or plant organs. The preferred aim upon use is the mediation of resistance to plant-enzyme inhibitors.
Depending on the choice of the promoter, expression can take place specifically in the leaves, in the seeds or in other plant organs. Such transgenic plants, their propagation material and their plant cells, plant tissues or plant organs are a further subject of the present invention.
The transfer of foreign genes into the genome of a plant is termed transformation. In this process, the above-described methods of transforming and regenerating plants from plant tissues or plant cells are utilized for transient or stable transformation. Suitable methods are protoplast transformation by polyethylene glycol-induced DNA uptake, the biolistic [sic] approach using the gene gun, electroporation, incubation of dry embryos in DNA-containing solution, microinjection and Agrobacterium-mediated gene transfer. The methods mentioned are described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, editors: S. D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus, Annu.Rev.Plant Physiol.Plant Molec.Biol. 42 (1991) 205-225). The construct to be expressed is preferably cloned into a vector which is suitable for the transformation of Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711).
Agrobacteria which have been transformed with an expression cassette according to the invention can then be used in the known manner for transforming plants, in particular crop plants such as cereals, maize, soya, rice, cotton, sugar beet, canola, sunflower, flax, potato, tobacco, tomato, oilseed rape, alfalfa, lettuce and the various tree, nut and Vitis species, for example by bathing wounded leaves or leaf sections in an agrobacterial solution and subsequently growing them in suitable media.
Functionally equivalent sequences which encode a herbicide-binding polypeptide are, in accordance with the invention, those sequences which still have the desired functions, despite a different nucleotide sequence. Thus, functional equivalents encompass naturally occurring variants of the sequences described herein, and also artificial nucleotide sequences, for example artificial nucleotide sequences which have been obtained by chemical synthesis and are adapted to the codon usage of a plant.
In particular, functional equivalent is to be understood as a natural or artificial mutation of an originally isolated sequence which encodes the herbicide-binding polypeptide, which mutation continues to show the desired function. Mutations encompass substitutions, additions, deletions, exchanges or insertions of one or more nucleotide residues. Thus, the present invention also encompasses those nucleotide sequences which are obtained by modifying this nucleotide sequence. The purpose of such a modification can be, for example, the further limitation of the encoding sequence contained therein, or else, for example, the insertion of more cleavage sites for restriction enzymes.
Other functional equivalents are those variants whose function is less or more pronounced, in comparison with the starting gene or gene fragment.
Moreover, artificial DNA sequences are suitable as long as they induce the desired resistance to herbicides, as described above. Such artificial DNA sequences can be identified, for example, by backtranslating proteins which have herbicide-binding activity and which have been constructed by means of molecular modeling, or by in vitro selection. Especially suitable are encoding DNA sequences which have been obtained by backtranslating a polypeptide sequence in accordance with the codon utilization which is specific to the host plant. The specific codon utilization can be determined readily by an expert familiar with methods of plant genetics by computer-aided evaluation of other, known genes of the plant to be transformed.
Further suitable equivalent nucleic acid sequences according to the invention which must be mentioned are sequences which encode fusion proteins, where part of the fusion protein is a non-plant-derived herbicide-binding polypeptide or a functionally equivalent part thereof. For example, the second part of the fusion protein can be a further polypeptide with enzymatic activity, or an antigenic polypeptide sequence with the aid of which detection of scFvs expression is possible (for example myc-tag or his-tag). However, it is preferably a regulatory protein sequence, for example a signal or transit peptide, which directs the polypeptide with herbicide-binding properties to the desired site of action.
However, the invention also relates to the expression products produced in accordance with the invention and to fusion proteins of a transit peptide and a polypeptide with herbicide-binding properties.
Resistance/tolerance means, for the purposes of the present invention, the artificially acquired ability of plants to withstand the action of plant enzyme inhibitors. It embraces the partial and, in particular, complete insensitivity to these inhibitors for the duration of at least one plant generation.
The primary site of action of herbicides is generally the leaf tissue, so that leaf-specific expression of the exogenous herbicide-binding polypeptide is capable of providing sufficient protection. However, one will understand readily that the action of a herbicide need not be restricted to the leaf tissue, but may also be effected in all remaining organs of the plant in a tissue-specific manner.
In addition, constitutive expression of the exogenous herbicide-binding polypeptide is advantageous. On the other hand, inducible expression may also be desirable.
The efficacy of the transgenically expressed polypeptide with herbicide-binding properties can be determined for example in vitro by shoot meristem propagation on herbicide-containing medium in series with staggered concentrations, or via seed germination tests. In addition, the herbicide tolerance, of a test plant, which has been altered with regard to type and level can be tested in greenhouse experiments.
The invention furthermore relates to transgenic plants, transformed with an expression cassette according to the invention, and to transgenic cells, tissues, organs and propagation material of such plants. Especially preferred are transgenic crop plants, for example cereals, maize, soya, rice, cotton, sugar beet, canola, sunflower, flax, potato, tobacco, tomato, oilseed rape, alfalfa, lettuce and the various tree, nut and Vitis species.
The transgenic plants, plant cells, plant tissues or plant organs can be treated with an active ingredient which inhibits the plant enzymes, whereby the plants, plant cells, plant tissues or plant organs which have not been transformed successfully die. Examples of suitable active ingredients are in particular 5-(2-chloro-4-(trifluoromethyl)phenoxy)-2-nitrobenzoic acid (acifluorfen) and 7-chloro-3-methylquinolin-8-carboxylic acid (quinmerac), and metabolites and functional derivatives of these compounds. The DNA which encodes a polypeptide with herbicide-binding properties and which has been inserted into the expression cassettes according to the invention can thus also be used as selection marker.
The present invention has the advantage, in particular in the case of crop plants, that, once a selected resistance of the crop plant to the plant enzyme inhibitors has been induced, such inhibitors can be employed as specific herbicides to the non-resistant plants. Herbicidal compounds from the groups bl-641 may be mentioned as examples of such inhibitors, but not by way of limitation:
b1 1,3,4-Thiadiazoles:
buthidazole, cyprazole
b2 Amides:
allidochlor (CDAA), benzoylprop-ethyl, bromobutide, chlorthiamid, dimepiperate, dimethenamid, diphenamid, etobenzanid (benzchlomet), flamprop-methyl, fosamin, isoxaben, monalide, naptalame, pronamid (propyzamid), propanil
b3 Aminophosphoric acids:
bilanafos, (bialaphos), buminafos, glufosinate-ammonium, glyphosate, sulfosate
b4 Aminotriazoles:
amitrole
b5 Anilides:
anilofos, mefenacet
b6 Aryloxyalkanoic acids:
2,4-D, 2,4-DB, clomeprop, dichlorprop, dichlorprop-P, dichlorprop-P (2,4-DP-P), fenoprop (2,4,5-TP), fluoroxypyr, MCPA, MCPB, mecoprop, mecoprop-P, napropamide, napropanilide, triclopyr
b7 Benzoic acids:
chloramben, dicamba
b8 Benzothiadiazinones:
bentazone
b9 Bleachers:
clomazone (dimethazone), diflufenican, fluorochloridone, flupoxam, fluridone, pyrazolate, sulcotrione (chlormesulone)
b10 Carbamates:
asulam, barban, butylate, carbetamid, chlorbufam, chlorpropham, cycloate, desmedipham, di-allate, EPTC, esprocarb, molinate, orbencarb, pebulate, phenisopham, phenmedipham, propham, prosulfocarb, pyributicarb, sulf-allate (CDEC), terbucarb, thiobencarb (benthiocarb), tiocarbazil, tri-allate, vernolate
b11 Quinoline acids:
quinclorac, quinmerac
b12 Chloroacetanilides:
acetochlor, alachlor, butachlor, butenachlor, diethatylethyl, dimethachlor, metazachlor, metolachlor, pretilachlor, propachlor, prynachlor, terbuchlor, thenylchlor, xylachlor
b13 Cyclohexenones:
alloxydim, caloxydim, clethodim, cloproxydim, cycloxydim, sethoxydim, tralkoxydim, 2-{1-[2-(4-chlorophenoxy)propyloxyimino]butyl}-3-hydroxy-5-(2H-tetrahydrothiopyran-3-yl)-2-cyclohexen-1-one
b14 Dichloropropionic acids:
dalapon
b15 Dihydrobenzofurans:
ethofumesate
b16 Dihydrofuran-3-ones:
flurtamone
b17 Dinitroanilines:
benefin, butralin, dinitramin, ethalfluralin, fluchloralin, isopropalin, nitralin, oryzalin, pendimethalin, prodiamine, profluralin, trifluralin
b18 Dinitrophenols:
bromofenoxim, dinoseb, dinoseb-acetate, dinoterb, DNOC
b19 Diphenyl ethers:
acifluorfen-sodium, aclonifen, bifenox, chlornitrofen (CNP), difenoxuron, ethoxyfen, fluorodifen, fluoroglycofen-ethyl, fomesafen, furyloxyfen, lactofen, nitrofen, nitrofluorfen, oxyfluorfen
b20 Dipyridylenes:
cyperquat, difenzoquat-methylsulfate, diquat, paraquat dichlorid
b21 Ureas:
benzthiazuron, buturon, chlorbromuron, chloroxuron, chlortoluron, cumyluron, dibenzyluron, cycluron, dimefuron, diuron, dymrone, ethidimuron, fenuron, fluormeturon, isoproturon, isouron, karbutilate, linuron, methabenzthiazuron, metobenzuron, metoxuron, monolinuron, monuron, neburon, siduron, tebuthiuron, trimeturon
b22 Imidazoles:
isocarbamid
b23 Imidazolinones:
imazamethapyr, imazapyr, imazaquin, imazethabenz-methyl (imazame), imazethapyr
b24 Oxadiazoles:
methazole, oxadiargyl, oxadiazon
b25 Oxiranes:
tridiphane
b26 Phenols:
bromoxynil, ioxynil
b27 Phenoxyphenoxypropionic esters:
clodinafop, cyhalofop-butyl, diclofop-methyl, fenoxapropethyl, fenoxaprop-p-ethyl, fenthiapropethyl, fluazifop-butyl, fluazifop-p-butyl, haloxyfop-ethoxyethyl, haloxyfop-methyl, haloxyfop-p-methyl, isoxapyrifop, propaquizafop, quizalofopethyl, quizalofop-p-ethyl, quizalofop-tefuryl
b28 Phenylacetic acids:
chlorfenac (fenac)
b29 Phenylpropionic acids:
chlorophenprop-methyl
b30 Protoporphyrinogen IX oxydase inhibitors:
benzofenap, cinidon-ethyl, flumiclorac-pentyl, flumioxazin, flumipropyn, flupropacil, fluthiacet-methyl, pyrazoxyfen, sulfentrazone, thidiazimin
b31 Pyrazoles:
nipyraclofen
b32 Pyridazines:
chloridazon, maleic hydrazide, norflurazon, pyridate
b33 Pyridinecarboxylic acids:
clopyralid, dithiopyr, picloram, thiazopyr
b34 Pyrimidyl ethers:
pyrithiobac-acid, pyrithiobac-sodium, KIH-2023, KIH-6127
b35 Sulfonamides:
flumetsulam, metosulam
b36 Sulfonylureas:
amidosulfuron, azimsulfuron, bensulfuron-methyl, chlorimuronethyl, chlorsulfuron, cinosulfuron, cyclosulfamuron, ethametsulfuron-methyl, ethoxysulfuron, flazasulfuron, halosulfuronmethyl, imazosulfuron, metsulfuron-methyl, nicosulfuron, primisulfuron, prosulfuron, pyrazosulfuron-ethyl, rimsulfuron, sulfometuron-methyl, thifensulfuron-methyl, triasulfuron, tribenuron-methyl, triflusulfuron-methyl
b37 Triazines:
ametryn, atrazine, aziprotryn, cyanazine, cyprazine, desmetryn, dimethamethryn, dipropetryn, eglinazine-ethyl, hexazinone, procyazine, prometon, prometryn, propazine, secbumeton, simazine, simetryn, terbumeton, terbutryn, terbutylazine, trietazine
b38 Triazinones:
ethiozin, metamitron, metribuzin
b39 Triazolecarboxamides:
triazofenamid
b40 Uraciles
bromacil, lenacil, terbacil
b4Others:
benazolin, benfuresate, bensulide, benzofluor, butamifos, cafenstrole, chlorthal-dimethyl (DCPA), cinmethylin, dichlobenil, endothall, fluorbentranil, mefluidide, perfluidone, piperophos
The spectrum of action of functionally equivalent derivatives of plant enzyme inhibitors is comparable with the spectrum of action of the substances named individually, while the inhibitory activity (for example expressed in g of inhibitor per hectare under cultivation, required for completely suppressing the growth of non-resistent plants) is lower, identical or higher.
The invention is now illustrated by the examples which follow, but is not limited thereto:
General Cloning Methods
The cloning steps carried out within the scope of the present invention, for example restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linkage of DNA fragments, transformation of E. coli cells, cultivation of bacteria, multiplication of phages and sequence analysis of recombinant DNA were carried out as described by Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6).
The bacterial strains used hereinbelow (E. coli, XL-I Blue) were obtained from Stratagene. The agrobacterial strain used for the transformation of plants (Agrobacterium tumefaciens, C58C1 with plasmid pGV2260 or pGV3850kan) was described by Deblaere et al. (Nucl. Acids Res. 13 (1985) 4777). Alternatively, the agrobacterial strain LBA4404 (Clontech) or other suitable strains may also be used. The vectors pUC19 (Yanish-Perron, Gene 33(1985), 103-119) pBluescript SK-(Stratagene), pGEM-T (Promega), pZerO (Invitrogen), pBin19 (Bevan et al., Nucl. Acids Res. 12(1984) 8711-8720) and pBinAR (Hxc3x6fgen and Willmitzer, Plant Science 66 (1990) 221-230) were employed for cloning purposes.
Sequence Analysis of Recombinant DNA
Recombinant DNA molecules were sequenced using a laser fluorescence DNA sequencing apparatus from Pharmacia, using the method of Sanger (Sanger et al., Proc. Natl. Acad. Sci. USA 74(1977), 5463-5467).
Generation of Plant Expression Cassettes
A 35S CaMV promoter was inserted into plasmid pBin19(Bevan et al., Nucl. Acids Res. 12, 8711 (1984)) in the form of an EcoRI-KpnI fragment (corresponding to nucleotides 6909-7437 of the cauliflower mosaic virus (Franck et al. Cell 21 (1980) 285). The polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984) 835), nucleotides 11749-11939, was isolated in the form of a PvuII-HindIII fragment and, after SphI linkers had been added, cloned into the PvuII cleavage site between the SphI-HindIII cleavage site of the vector. This gave plasmid pBinAR (Hxc3x6fgen and Willmitzer, Plant Science 66 (1990) 221-230).